All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Electrochemiluminescence is a technology having broad application in the fields of diagnostics, drug discovery and basic research. The technology has particular utility in the design of immunoassays, which benefit from increased sensitivity, dynamic range, kinetics and format flexibility by electrochemiluminescence technology. In particular, incubation times may be significantly reduced, due to the solution phase kinetics used and the elimination of wash steps (1).
Electrochemiluminescence is the process by which light generation occurs when low voltage is applied to an electrode, triggering a cyclical oxidation and reduction reaction of a heavy metal ion, such as ruthenium. The heavy metal ion is bound in a chelate of tris-bipyridine (i.e., Ru(bpy).sub.3.sup.2+). A second reaction component is an electron carrier, such as tripropylamine (TPA), which mediates the redox reaction. This electron carrier is present in excess, and is consumed during the assay while the heavy metal chelate is recycled. Because the metal chelate is recycled and the carrier is present in excess, the signal generated from the assay is intensified (1, 2).
The luminescent reaction is triggered upon application of an electric potential. There are currently several instruments available on the market designed to deliver the necessary voltage and quantify the resulting signal. These products include ORIGEN.RTM. (Igen Corp.) and QPCR System 5000.RTM. (Perkin-Elmer Corp.) magnetic electrochemiluminescence detectors (2). The ORIGEN.RTM. instrument operates using magnetic beads which have been designed to interact with or bind to the antigen of interest. The beads are added to the reaction tubes and the tubes are placed in a vortexing carousel which keeps the beads in suspension prior to sampling. An automatic sample delivery system aspirates a user-determined volume of each reaction mixture and pumps it into the flow cell. As the sample is pumped through the cell the magnetic beads are captured by a magnet onto a platinum electrode which delivers the required electric potential. The light subsequently emitted is measured in a photomultiplier tube and digitally recorded. The cell is then washed in preparation for the next reading (1).
In recent years, many papers have been published reporting the success of magnetic electrochemiluminescent techniques in the detection and quantitation of various targets, including bacteria (3), biotoxoids and bacterial spores (2), specific antigens and macromolecules (4-6), and nucleic acids (5, 7). However, no assays or diagnostic applications have been reported which apply immunomagnetic electrochemiluminescence (IM-ECL) to eukaryotic cells. Yu and Bruno suggest that IM-ECL technology is broadly applicable, even to eukaryotic cells, and make reference to an early publication by researchers at Igen Corporation (3). However, a review of the referenced publication reveals no such disclosed use (5). Moreover, there are no other reports of such a use in the literature.
The absence of reported IM-ECL assays using eukaryotic cells is probably due to the vortexing step typically employed by IM-ECL instruments which keeps the samples in suspension until an aliquot is aspirated for quantitation. Although IM-ECL technology has been used to detect bacterial cells (3), it has long been presumed by those of skill in the art that eukaryotic cells, and in particular animal cells, were simply too fragile to be used in such assays.
The present inventors have surprisingly found that IM-ECL instrumentation may be employed to conduct immunoassays wherein eukaryotic cells are targeted. This discovery allows further expansion of this helpful technology to cover new assay formats, and should be applicable to all assay types, including competitive assays and detection/ diagnostic techniques.